In the earlier research efforts conducted by one of the present inventors, a group of recombinant bacterial plasmid cloning vehicles for expression of exogenous genes in transformed bacterial hosts was constructed, comprising a DNA insert fragment coding for the desired polypeptide, linked in reading phase with one or more functional fragments derived from any outer membrane protein gene of any gram-negative bacterium. In a preferred embodiment of these expression plasmids, the exogenous DNA codes for mammalian hormones, enzymes or immunogenic proteins (or intermediates therefor), the functional fragments are derived from the lipoprotein gene of E. coli, and the desired polypeptide is expressed in E. coli transformants. In a more preferred embodiment, the DNA sequence coding for the desired protein is linked with and is expressed under the control of four specific functional fragments associated with the E. coli lipoprotein gene, namely, the promoter, the 5'-untranslated region, the 3'-untranslated region and the transcription termination site of that gene.
These expression plasmids may also include a second promoter, preferably an inducible promoter and most preferably a DNA sequence consisting of 95 base pairs ("bp") and containing the E. coli .beta.-galactosidase or "lac" promoter-operator, which is inserted immediately downstream of the lipoprotein promoter. This region serves not only as another transcription initiation site, but also as a repressor-binding site, thus working as a transcriptional "switch" for transcription initiated from the lipoprotein or "lpp" promoter, so that the exogenous DNA is expressed only in the presence of a "lactose inducer." When induced, the DNA coding for the desired polypeptide is transcribed from both promoters, thereby increasing the yield of the desired product over that obtained when expression is directed by the inducible lac promoter-operator alone. Accordingly, either constitutive or inducible gene expression may be achieved using these lpp gene cloning vehicles, provided that when inducible expression is desired, special E. coli strains are used as transformants, specifically, those which carry a mutant gene which overproduces the lactose repressor molecule.
Most preferably, each class of expression plasmids (both constitutive and inducible) includes three subclasses of plasmids, the members of each subclass containing one of three alternative insertion sites. In this manner, the selection of a particular plasmid or a particular sub-class of plasmids for gene expression can influence the ultimate location at which the expression product can be found and collected. Using one of these insertion sites, for example, the desired polypeptide can be expressed with a leader sequence located at the amino terminal, the leader comprising the signal peptide of the E. coli lipoprotein, such that the desired product may be secreted through the cytoplasmic membrane and the signal peptide removed in vivo by processes native to the transformant, to yield the exogenous gene product. On the other hand, using expression plasmids which contain one or the other of the two remaining insertion sites, the expression product can be expected to be found either in the cytoplasm of the cell, or in the cell wall.
While the plasmids of each sub-class share a common insertion site, they differ from one another in their individual reading frames. Thus, each sub-class comprises three plasmids, whose reading frames in effect differ by one base pair, enabling the selection of any desired reading frame for each insertion site and thereby facilitating the use of these expression vehicles with a wide variety of DNA insert fragments without the necessity of any direct modification of the reading frames of those fragments.
The earlier research efforts of one of the present inventors also provided a modification of the foregoing scheme, namely, a class of "auto-regulated" inducible expression cloning vehicles, each member of the class being otherwise identical with its analog in the class of inducible expression vehicles described above, but further including the DNA sequence coding for the repressor molecule capable of binding with the inducible promoter used in the vehicle. In the preferred embodiment, the autoregulated expression plasmids incorporate an intact, functional E. coli lacI gene for this purpose. Since the expression of the exogenous information in these cloning vehicles is regulated from within each cloning vehicle itself, a more effective transcriptional "switch" is provided, insuring that transcription is completely repressed in the absence of the appropriate inducer, without the necessity of utilizing special E. coli strains which overproduce the repressor molecule as transformants.